Protein A from Staphylococcus aureus binds with high affinity and high specificity to the C.gamma.2-C.gamma.3 interface region of IgG (Langone, Adv. Immunol., 32: 157-252 (1982)). Protein A also exhibits an affinity for the Fab region of immunoglobulins that are encoded by the V.sub.H gene family, V.sub.H III (Sasso et al., J. Immunol, 61: 3026-3031 (1991); Hillson et al., A. J Exp. Med., 178: 331-336 (1993)). The sequence of the gene coding for protein A revealed two functionally distinct regions (Uhlen et al., J. Biol. Chem., 259: 1695-1702 (1984); Lofdahl et al., Proc. Natl. Acad. Sci (USA), 80: 697-701 (1983)). The amino-terminal region contains five highly homologous IgG-binding domains (termed E, D, A, B and C), and the carboxy terminal region anchors the protein to the cell wall and membrane. All five IgG-binding domains of protein A bind to IgG via the Fc region.
The structure of the B domain has been studied using .sup.1 H-NMR (Torigoe et al., Biochem, 29: 8787-8793 (1990); Gouda et al, Biochem., 31: 9665-9672 (1992)) and found to consist of three .alpha.-helical regions (.alpha..sub.1, .alpha..sub.2, .alpha..sub.3 corresponding to helices I, II and III in the NMR structure) which also are retained when bound to the Fe region of IgG (Gouda, supra). The tri-helical nature of the bound state is in contrast to the X ray crystal structure reported by Deisenhofer, Biochem., 20: 2361-2370 (1981) which showed that .alpha..sub.1 and .alpha..sub.2 helices of the B domain were present while the .alpha..sub.3 helix did not form. The X ray crystallographic analysis reported by Deisenhofer, supra, and the mutagenesis studies reported by Popplewell et al., Protein Eng., 4: 963-970 (1991) and Cedergren et al., Protein Eng., 6: 441-448 (1993) have also identified ten residues within .alpha..sub.1 and .alpha..sub.2 and nine residues within the Fc region of IgG which participate in the protein--protein interaction.
The interaction between protein A and IgG forms the basis of many immunoaffinity-based purification procedures for antibodies (Hjelm, FEBS Lett., 28: 73-76 (1972)), antibody fragments and antigens (Sisson and Carter, Immunol. Meth., 127: 215-22-(1990)). Bottomley et al., J. Immunol. Meth., 182: 185-192 (1995) reported that truncating B domain variant peptides lacking the 13 C-terminal residues of the .alpha..sub.3 helix causes a decrease in IgG-binding activity. Bottomley et al. also characterized various mutations of residues within the .alpha..sub.1 and .alpha..sub.2 regions found to reduce or not affect IgG-binding activity.